Women normally have more body fat, a different distribution of fat, and a greater prevalence of obesity than men. Despite their greater body fat, premenopausal women are less prone to develop insulin resistance diseases associated with obesity. Therefore, an understanding of gender differences in fat metabolism has important physiological and clinical implications. However, few studies have evaluated the effect of gender on in vivo regulation of fat metabolism in humans. Moreover, the available data is largely confounded by differences in body composition, which independently influence substrate metabolism. The main purpose of this proposal is to determine the independent effects of gender and adiposity on fat metabolism during the major physiological conditions which decrease lipolysis and fat oxidation (hyperinsulinemia) or increase the mobilization and use of fat as a fuel (fasting and endurance exercise). We hypothesize that differences in fat metabolism between men and women will become more apparent during the physiological challenges of hyperinsulinemia, exercise, and fasting. The proposed studies will elucidate the independent effects of gender and adiposity in men and premenopausal women, matched by adiposity at three levels (22-25%, 28-31%, and 35-38% body fat), on: 1) insulin action in different tissues: adipose tissue (suppression of whole- body and regional adipose tissue lipolysis), liver (inhibition of glucose production), and skeletal muscle (stimulation of glucose uptake) (Study 1); the metabolic responses to short-term fasting (whole-body and regional adipose tissue lipolysis and its hormonal regulation) (Study 2); and 3) the metabolic responses to endurance exercise (whole-body and regional adipose tissue lipolysis, intramuscular triglyceride lipolysis, whole body fat oxidation, plasma fatty acid oxidation, and intramuscular triglyceride oxidation rates) (Study 3). These endpoints will be evaluated by performing isotope infusion experiments during 1) a multistage hyperinsulinemic pancreatic hormonal clamp, 2) short-term fasting, and 3) cycling exercise using state-of-the-art in vivo methodologies, including stable isotope tracers to measure substrate and norepinephrine kinetics, indirect calorimetry to evaluate substrate oxidation, microdialysis and abdominal vein catheterization to assess regional (abdominal/femoral adipose tissue and skeletal muscle) substrate and hormone metabolism, and 133Xe clearance and venous occlusion plethysmography to measure adipose tissue and muscle blood flow, respectively. The information derived from these studies may improve our understanding of the regulation fat metabolism and the underlying metabolic factors which lead to obesity-related comorbidities.